The long-term objective of this grant proposal is to understand how the ribosome functions at the atomic level during protein synthesis. Specific goals include developing a genetic system in the extreme thermophile Thermus thermophilus to construct and isolate mutants in rRNA and ribosomal proteins. Mutant ribosomes which form alternate conformations in the translation cycle will be crystalized (in collaboration with Drs. Jamie Cate and Venki Ramakrishnan). A second goal is to investigate dynamic aspects of E. coli ribosomes by utilizing a series of genetic and biochemical methods developed in our laboratory over several years. The primary approach involves the construction of rRNA mutations in a plasmid-borne rrn operon. A variety of strains and vectors permit even lethal mutations to be isolated. The atomic coordinates provided by crystal studies of the 30S and 50S subunits will be used in designing mutagenic strategies to probe ribosome structure and function during peptide bond formation, translocation and decoding. Mutants forming stable functional intermediates will be characterized by a variety of functional assays and studied by cryoEM (in collaboration with Dr. Joachim Frank). Some of these mutants will be constructed and expressed in Thermus thermophilus for crystal studies. A third goal involves the application of these same methods to probe the mechanism of action and resistance of antibiotics affecting protein synthesis. These studies should provide fundamental insights about ribosome function and conformational changes in the rRNA and ribosomal proteins involved in tRNA selection, translocation, peptide bond formation and signal transmission within and between the subunits.